Construction Of Two Expression Shuttle Vectors And A Surface Display Vector For Bacillus Subtilis

As a bacterial host generally recognized as safe either for efficiently expressingheterogeneous protein or being a whole cell enzyme or a carrier of oral vaccine,comparing with many other bacteria, Bacillus subtilis is widely researched andapplied to industrial fermentation. Although there are many plasmids which have beenused for exogenous protein expression in B. subtilis, the E. coli-B. subtilis expressionshuttle vector charactered with convenience in genetic munipulation, high efficientexpression and exogenous protein directly displaying on spore surface are still lack.In this study, we constructed two expression shuttle vectors and a surface displayvector for B. subtilis and verified their ability using four reporter genes. The maincontents are as follows:1. A DNA fragment containing the sequence of xylose promoter (PxylR) from B.subtilis and the sequence of T7peptide and multiple cloning sites from a Escherichiacoli expression vector pET21b were amplified by fusion PCR. After that, an E. coli-B.subtilis expression shuttle vector pTL, which can be induced exogenous proteinexpression by D-xylose, was constructed by inserting the fusion DNA fragmentinstead of the multiple clone site of the shuttle vector pHT315. The genes gfp, gus,APPsw, lipa encoding green fluorescent protein (GFP), β-glucuronidase (GUS),amyloid precursor protein (APPsw) and lipase (from Serratia marcescens) separately,were amplified by PCR and inserted into the multiple cloning site of the vector pTLfor construction of four recombinant plasmids. The recombinant plasmids weretransformed into B. subtilis WB800and obtained the engineered strains WB800-gfp,WB800-gus, WB800-APPsw and WB800-lipa. The green fluorescence can beobserved in WB800-gfp cells detected by fluorescence microscope, which indicatedthat GFP was successfully expressed. The optimal excitation and emission wavelengthof GFP, were390nm and507nm, was detected and determined by fluorescencespectrophotometry. Using gfp as a report gene, the preliminary optimal condition forGFP expression induced by D-xylose in WB800-gfp was as follows: The highest GFPexpression was reached by adding1%D-xylose as the final concentration and theculture was incubated at37oC,200rpm for8h. The expression of GUS, APPsw andLipA were induced at this condition. That the color strain of WB800-gus turned blueimplied the positive reaction. Western blot test result showed that the cell lysate ofWB800-APPsw had positive protein band at85kDa. The lipase activity ofWB800-lipa cell lysate was72.73±0.25U/mg assayed with Rashid N p-NPPcolorimetry. The lipase activity was increased nearly210times comparing with that of WB800-lipa without xylose induction. These results indicated that these three geneswere highly expressed. The plasmid stability test revealed that pTL showed98.5%stability up to10generations. All the results above demonstrate that pTL is an E.coli-B. subtilis expression shuttle vector with characters of easy to handle, highlyefficient expression as well as high stability. pTL will facilitate the expression of theforgein protein as a fusion protein with T7peptied which almost has no effect on theproperties of the forgein protein and benefit the subsequent purification.2. An E. coli-B. subtilis shuttle vector pDG150containing Pspac promoter wasconstructed on the basis of pDG148-stuI. The genes gfp, gus, APPsw and lipa wereinserted into pDG150to construct the recombinant plasmids. The recombinantplasmids were transformed into B. subtilis168and the engineered strains B168-gfp,B168-gus, B168-APPsw and B168-lipa were generated. The results detected byfluorescence microscopy and fluorescence spectrophotometer indicated that the GFPin B168-gfp was highly expressed. Using gfp as report gene, we obtained thepreliminary optimal condition induced by IPTG according to the expression level ofGFP in B168-gfp using the fluorescence spectrophotometry assay method. Thecondition for the highest expression of GFP was obtained by1mM IPTG inductionfor4h at37oC,200rpm. Under the above condition, the expression of GUS inB168-gus was positive assayed by GUS staining method; Western blot result revealedthat the cell lysate of B168-APPsw had the positive protein bands at78kDa, whichindicated that APPsw had expressed; The lipase was expressed in B168-lipa provedby Rashid N p-NPP colorimetry method and the lipase activity induced by IPTG wasup to20.52±0.12U/mg which increased nearly50times comparing to the untreatedcontrol. The stability rate of plasmid pDG150was95.5%after continuously culturedfor10generations.3. A surface display vector pDG150-cotg was constructed by recombination of theplasmid pDG150and cotg gene including the promoter and the open reading frame(without the stop codon) of the spore coat protein CotG. The genes gfp, gus, APPswand lipa were inserted into pDG150-cotg to generate the recombinant plasmids. Theseplasmids were transformed into B. subtilis168and the engineered strains G168, U168,A168and L168were obtained. The spores of the engineered strains were harvestedafter cultured in DSM medium at37oC,200rpm for40h. GFP had successfullydisplayed on the spores of G168, which proved by fluorescence microscopy. Thespores of U168showed the positive color after detected by GUS staining, whichindicated GUS had expressed; Western Blot test showed that the spore extracts fromA168had positive protein bands in size of102kDa, indicating that APPsw proteinhad successfully displayed on the spores of A168which facilitated the preparation ofAPPsw antibody; The lipase activity was28±1.32U/g detected on the spores ofL168using Rashid N p-NPP colorimetry assay method.